Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

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In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

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Many pharmaceuticals are excreted in wastewater as parent substances or metabolites subsequent to therapeutic or diagnostic application in medical care. This includes the antiepileptic carbamazepine, which is not removed during conventional wastewater treatment and was found to be ubiquitous in the aquatic environment. Some carbamazepine metabolites have also been found in treated wastewater, but only five of them have been studied to date. However, at least 30 carbamazepine metabolites have been identified in humans, including some pharmacologically active or genotoxic compounds. Oxcarbazepine, an antiepileptic which is increasingly used, generates metabolites common to those of carbamazepine. The present work focuses on the presence of carbamazepine, oxcarbazepine, and seven of their metabolites (carbamazepine-10,11-epoxide, 10-hydroxy-10,11-dihydrocarbamazepine, 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, 2-hydroxy-carbamazepine, iminostilbene, acridine, and acridone) at three different treatment plants (conventional activated sludge, trickling filter, and stabilization ponds) selected in France. The main aim of this work was to identify selected compounds in wastewater after therapeutic use and to measure concentrations in influents and effluents at the three wastewater treatment plants. Except for iminostilbene, all of these compounds were detected in wastewater. The metabolite common to carbamazepine and oxcarbazepine, i.e., 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, was detected at a higher concentration than the parent substances in wastewater. The presence of parent molecules was noted in inlet and outlet water samples. Carbamazepine, as expected, was not removed by conventional activated sludge treatment. Nevertheless, in a wastewater treatment plant with a 78-day hydraulic retention time, a 73% decrease in carbamazepine concentration was observed. For the first time, oxcarbazepine was found in environmental samples. A decrease in oxcarbazepine concentrations was observed at the three sewage treatment plants, with removal ranging from 24 to 73%. No metabolite removal was observed after activated sludge treatment. In the two other sewage treatments plants, the fate of the metabolites differed. The concentration of some metabolites, e.g., 10,11-dihydro-10,11-trans-dihydroxycarbamazepine and acridine, increased, possibly via different processes such as cleavage of glucuronide conjugates or biotic and abiotic degradation of parent compounds. The behavior of the studied substances is discussed in terms of the treatment process and hydraulic retention time.